HIV-1 encodes proteins that modulate the host cellular environment to optimize viral replication and avoid host defenses. The accessory protein Vpu accomplishes this in part by antagonizing the activity of the host protein BST2 (also known as HM1.24, CD317, and tetherin). BST2 is an interferon-inducible integral membrane protein. This protein is the restriction factor that Vpu counteracts to stimulate the release of virus particles (virions) from infected host cells. We and others have shown that BST2 directly holds nascent virions to the plasma membrane of infected cells; that Vpu counteracts this by removing BST2 from its site of action at the plasma membrane; that BST2 is upregulated in response to HIV replication in vivo; and that BST2 serves a signaling and virus-sensing function through the induction of NF-kB transcriptional activity. The latter finding fits an emerging paradigm in which retroviral restriction factors have multifaceted roles during the innate immune response. Moreover, the field has developed evidence that HIV-1 Vpu specifically adapted to acquire activity as an antagonist of BST2 upon transmission of SIVcpz from chimpanzees to humans. Several key questions now need to be answered, and these are the basis for the specific aims of the proposal: 1) what are the molecular determinants and topologies of the protein's restricting and signaling activities? 2) in addition to the TrCP containing ubiquitin ligase complex, what are the cellular cofactors that support the antagonism of BST2 and how does Vpu interact with them? 3) how does BST2 activate NF-?B and detect viral gene expression? Does BST2 facilitate aspects of the adaptive immune response? And 4) does Vpu-mediated antagonism of BST2 contribute to HIV-1 transmission? Through the work proposed here, we expect to learn how the tethering and signaling functions of BST2 are structurally integrated; how Vpu modulates BST2 to antagonize restriction and signaling; the mechanisms by which BST2 signals and responds to viral gene expression and assembly; whether BST2 facilitates adaptive immunity; and whether any of the Vpu functions related to BST2 are optimized in HIV-1 variants that are transmitted between individuals. We expect to support or reject the notion that BST2-antagonism is an important aspect of HIV-1's ability to escape immune surveillance and establish a persistent infection in the human host.